Method of preparing 6-amino-5-hydroxy-1, 3-cyclohexadiene-1-carboxylic acid



Sets

This invention relates to the preparation of 6-amino- S-hydroxy-1,3-cyclohexadiene-l-carboxylic acid of the formula:

C O OH The above compound is a useful intermediate for the preparation of various benzoic acid derivatives such as, for example, anthranilic acid, 2-amino-3-hydroxy benzoic acid, and the like. The novel compound may be converted to anthraniic acid simply by heating in concentrated hydrochloric acid. As is well known, anthranilic acid is a valuable intermediate used in the preparation of indigo. The novel compound may also be converted to 2-amino-3-hydroxybenzoic acid by dehydrogenation in boiling water over a palladium catalyst. 2-amino-3-hydroxybenzoic acid is a biologically active nicotinic acid precursor [.T.A.C.S. 70, 1847 (1948)].

The novel compound is produced by aerobically fermenting an aqueous nutrient medium with certain mutant strains of microorganisms of the species Streptomyces aureofaciens. A particularly preferred method involves the use of a new strain of S. aureofaciens which we have designated as S652, but as will be apparent from the detailed description hereinafter certain other strains of S. aureofaciens may also be used to produce this compound with varying degrees of effectiveness.

The new strain is a member of the species S.'Z1LII'0- faciens since it is a direct descendant of the chlortetracycline-producing strain of S. aureofaciens, A377, which was isolated from the soil and is described in the United States patent to Duggar No. 2,482,055, and which'is deposited at the Northern Regional Research Laboratories, Peoria, Illinois, as NRRL 2209. Mutagenic agents and selective agents used in obtaining this new strain include ultraviolet irradiation, nicotine and nitrogen mustard treatments, and phage exposure. Spontaneous mutation of chlortetracycline-producing strains of S. aureofaciens may also result in strains which will produce the new compound of this invention.

The new strain of S. aureofaciens possesses the same general characteristics as do the strains which produce the tetracyclines and differs in the same general manner that the tetracycline-producing and chlortetracycline-producing strains of S. aureofaciens differ from each other, as has been described in a number of scientific papers which have been published. The data appearing below will serve to illustrate the variation of Strain S652 from the original A377 strain available as NRRL 2209.

Szrepzomyces aureofaciens Strain S652 was differentiated from Streptomyces aureofaciens Strain A377 (NRRL 2209) by observation of growth characteristics on various media incubated at 26.5 C.

tent Q P 3,069,326 Patented Dec. 18, 1962 (1) GLYCEROL ASPARAGINE BEEF EXTRACT AGAR Percent Glycerol 1.0 L-asparagine 0.05 Beef extract 0.2 KH PO 0.05 Bacto agar 1.5 Distilled water, q.s. 100.0 percent. Ad ustment with 50% KOH to pH 7.0. Post sterilization, pH 7.2.

Streptomyces aureofaciens Strain S652 Strain A377 Growth Good to abundant, Good.

hyaline, white to apigmentous. Aerlal hyphae Abundant Slight to fair, white to light gray. Sporulatton Algundant, fawn 1 to Light gray. eaver. Diffusible pigment.-- Light olive yellow-.." Light yellow. Reverse White, hyaline Yellialw to light orangeye ow.

Color Harmony Manual, third edition, Container Corporation of America.

(2) DEXTRIN CZAPEK-DOX AGAR Percent Dextrin 1.0 NaNO 0.2 K HPO 0.1 MgSO .7H O 0.05 KCl 0.05 FeSO .7H O s 0.001 Bacto agar 1.5

Distilled water, q.s. 100.0 percent. Post sterilization, pH 7.2.

Strepto myces aureofaciens Strain S652 Strain A377 Growth Thin, entire, semi- Good.

opaque, white. Trace, white Aerial hyphae Abundant, mouse gray 1 to lead gray 1 Water-white surface globules.

Profuse.

Trace, pale yellow.

Aprgmentzous, pink trace.

Sporulation D1 tl'usible pigment. Reverse 1 Color Harmony Manual, third edition, Container Corporation of Tap water, q.s. 100.0 percent. Post sterilization, pH 6.5.

Streptomyces aurcofacicns Strain S652 Strain A377 Growth Excellent.

Abundant, fawn.

Excell ent, white to nude Sporulation AbundantJawnWobea- Profuse, uniform.

. ver. Soluble pigment Tawny to light amber Light yellow to amber. Reverse White to nude tan Light tan.

1 Color Harmony Manual, third edition, Container Corporation of America.

(4) OTHER MEDIA Streptomyccs aureofacicns Medium Strain S652 Strain A377 Nutrient agar-.. Sparse growth; hyaline Good growth. No aerial to semiopaque white. hyphae. Reverse: pale No aerialhyphae. Reyellow. Pale yellow verse: White. No solsoluble pigment. uble pigment.

Glucose aspara- Fair growth: white. Good growth. Aerial gine meat 'ex- Sparse aerial mycelhyphaewhite becoming tract agar. ium: White becoming increasingly gray with brown to rose taupe. increase with spore R e v e r s e: w h i t e. formation. Reverse:

Waksmans agar.

Potato slants Light yellow soluble pigment. Sporulation: sparse.

Fair to good growth:

Chartreuse tint. Aerlight yellow to pinkorange. Tracezyelloworange soluble pigment. Good growth. Aerial hyphae fair becoming ial mycelium: sparse abundant: White to to abundant iawn. taupe brown. Re- Sporulation: sparse to verse: Camel 1 to adobe abundant. Reverse: brown. Light yellow Hyaline chartreuse soluble pigment. tint. Light yellow soluble pigment.

Excellent, smooth, Profuse, moist, smooth moist, modulated modulated growth: growth: Light fawn 1 Light brown yellow to beige. Trace to beige to cedar. white aerial mycel- No soluble pigment. ium. No soluble pigment. 1 Purple milk No observable change... Slight white to pale yellow growth collar. Little significant pH change nor apparent peptonization in 14 days.

1 Color Harmony Manual, third edition, Container Corporation of America.

V28, S609, and S77. To determine whether a selected colony will produce the new compound, it is necessary merely to cultivate a strain of S. aureofaciens in a fermentation flask and at the end of the fermentation period, filter the mash and examine the neutral filtrate spectrophotometrically for ultraviolet absorption at 278-280 m characterizing the product in terms of its extinction coeificient value (Elf of 570 The conditions of the fermentation with the new strain of S. aureofaciens of this invention as well as the other strains referred to above are generally the same as those presently known for cultivating Streptomyces for the production of antibiotics or other products. Thus the fermentation medium contains the usual nutrients and mineral substances. Suitable nutrient substances include starch, dextrose, cane sugar, glucose, molasses, soybean meal, peanut meal, yeast, meat extracts, peptone, ammonium sulfate, urea, corn steep liquor, distillers solubles,- fish meal and other conventional substances. The inor' ganic salts include calcium carbonate, ammonium sulfate, ammonium chloride, and the various trace elements such as manganese, cobalt, zinc, copper, iron and the like.

In general, the fermentation is carried out for about 48 to 200 hours and at temperatures ranging from about 2040 C. The pH may range from 5.5-7.5.

The new compound may be isolated from the fermentation mash in any suitable manner. A preferred procedure involves an ion exchange separation in which the fermentation mash is filtered, and the filtrate is acidified to pH 1 to 2 with a suitable mineral acid such as hydrochloric acid, sulfuric acid, etc. The acidified aqueous filtrate is passed through an Amberlite IR-120 cation exchange resin column and the column is developed in the usual manner with an aqueous mineral acid, i.e., normal sulfuric acid. The eluted fraction is made alkaline by the addition of solid barium hydroxide with stirring. Barium sulfate precipitates and is filtered off while the pH of the aqueous solution is above pH 12. The filtrate is then neutralized with sulfuric acid and is concentrated in vacuo. Crystallization of the crude product occurs rapidly at room temperature (25 i5 C.). The product may then be purified in a standard manner by recrystallization from glacial acetic acid.

(5) MICROSCOPIC OBSERVATIONS Streptomyces aureofaciens Medium Strain S652 Strain A377 Myeelium Spores Mycelium Spores Glycerol asparagine meat Flexuous, continuous branched. spheroidal to ovoidal. Flexuous, continuous branched. Spheroidal to ovoidal.

extract agar. Dlam. 0.5-1.5;4. Diam. 0.5-1.0;4. Diam. 1.0-1.2;1. Diarn. 1.2-1.5

A1 4 eornsteep agar...-. Flexuous continuous branched. spheroidal to ovoidal. Flexuous, continuous branched. Sphcroidal to ovoidal.

Dram. 0.5-0.7q. Diam. 0.5a. Diam. 0.8- 1.0 Diam. 1.2-1.5;r.

Waksmans agar Flexucus, continuous branched. spheroidal to ovoidal. Flexuous, continuous branched. spheroidal to ovoidal.

Dram. 0.5-1.0p. Diam. 0.5l.0p. Diam. 0.5-1.0 Diam. 0.5-1.0;r.

Nora-Mycelial and spore morphology of Streptomyces aureofaciens Strain S652 is apparently similar to that of the original Strain A377.

Viable cultures of mutant S. aureofaciens Strain S652 which produce the new compound have been deposited with the American Type Culture Collection in Washington, DC, where. this strain has been assigned ATCC Accession Number 13,189.

It isto be understood that the invention is not limited to the use of Strain S652 for the production of the novel compound herein as other strains of S. aureofaciens may also be used with varying degrees of eifectiveness for the production of this compound as is indicated hereinabove. Thus we have used successfully strains designated as B740 (ATCC 12554), 8730-6, 8730-14, V11,

The invention will be described in greater detail in conjunction with the following specific examples.

Example 1 A typical medium used to grow the primary inoculum is prepared according to the following formulation:

Water to 1,000 milliliters.

Aliquots of this inoculum medium are placed in 8 inch test tubes which are sterilized at 120 C. and pounds pressure for minutes. Spores of a strain of S. aureofaciens capable of producing 6-arnino-5-hydroxy-l,3-cyclohexadiene-l-carboxylic acid, such as Strain S652, are

washed from an agar slant with sterile distilled water to form a suspension containing approximately 60x10 spores per milliliter. A 0.33 milliliter portion of this suspension is added to eachS-inch test tube containing the sterilized medium. These inoculated tubes are incubated for 24 hours at 28 C. on a reciprocating shaker operating at 116 oscillations per minute.

Example 2 A typical synthetic medium which may be used in the fermentation process employing strains of S. aureofaciens to produce the new compound is prepared according to the following formulation:

Water to 1,000 milliliters.

A 25 milliliter aliquot of this fermentation medium and 0.50 milliliters (2.0%) of lard oil are placed in a series of 250 milliliter Erlenmeyer flasks which are then sterilized at 120 C. and 15 pounds per square inch pressure for 20 minutes. The Erlenmeyer flasks are then inoculated with 1.0 milliliter of inoculum of S. aureofacieizs S652 prepared according to the procedure of Example 1, and incubated at 27 C. for 96 hours on a rotary shaker operating at 180 revolutions per minute. The mash is assayed spectrophotometrically and is found to contain 3,200 micrograms per milliliter of 6-amino-5- hydroxy-l,3-cyclohexadiene-1-carboxylic acid.

Example 3 25 milliliter aliquots of the synthetic fermentation medium shown in Example 2 and 0.50 milliliters (2.0%) of lard oil are placed in a series of 250 milliliter Erlenieyer flasks. These flasks are sterilized at 120 C. and 15 pounds per square inch pressure for 20 minutes. Following the sterilization procedure, 1.0 milliliter of inoculum of a strain of S. aureofaciens capable of producing G-amino-S-hydroxy-1,3-cyclohexadiene l carboxylic acid, prepared according to the procedure of Example 1, is added to each flask and the flasks are incubated at 27 C. for 96 hours on a rotary shaker operating at 180 revolutions per minute. The mask is assayed spectrophotometricaliy and is found to contain 3,900 micrograms per milliliter of 6-amino-5-hydroXy-1,3-cyclohexadiene-l-carboxylic acid.

Example 4 A fermentation medium of the following formulation is prepared:

Corn starch q. grams 55.0 Corn steep do 30.0 Cotton seed meal do 2.0 CaCO -do 7.0 (PIE-1492504 do NH Cl do 1.5 FeSO .7H O milligrams 40.0 MnSO .4H O do 50.0 ZnSO .7H O do 100.0 CoCl .6H O do 5.0

Water to 1,000 milliliters.

A 25 milliliter aliquot of this fermentation medium and 0.625 milliliter (2.5%) of lard oil are placed in a series of 25 0 milliliter Erlenmeyer flasks which are then sterilized at 120 C. and 15 pounds per square inch pressure for 20 minutes. The Erlenmeyer flasks are then inoculated with 1.0 milliliter of inoculum of S. aureofaciens Strain S652 prepared according to Example 1, and the flasks are incubated at 27 C. for 120 hours on a rotary shaker operating at 180 revolutions per minute. This mash is assayed spectrophotometrically and is found to contain 7 350 micrograms per milliliter of 6-amino-5-hydroxy-l,3- cyclohexadiene-l-carboxylic acid.

Example 5 The procedure of the preceding example is repeated except that the temperature is maintained at 37 C. with a 6-arnino-5-hydroxy-l,3-cyclohexadiene l carhoxylic acid producing strain of S. aureofaciens. At the end of the fermentation, the mash is assayed spectrophotometrically and is found to contain 5050 micrograms per milliliter of 6-amino-5-hydroxy-1,3-cyclohexadiene-l-carboxylic acid.

- Example 6 Ten liters of a fermentation mash prepared as in Example 4 is filtered and the pH of the filtrate is adjusted to pH 1.5 with concentrated hydrochloric acid. Spectrophotometric analysis of this filtrate shows 6.5 milligrams of 6-amino-5-hydroxy-1,3-cyclohexadiene 1 carhoxylic acid per milliliter of filtrate. This acidified mash filtrate is put through a 3-inch diameter column containing 3 kilograms of Amberlite 13-120 (H-form) cation exchange resin. The resin column is then washed with 15 liters of distilled Water. The column is eluted to extract the activity with 20 liters of normal sulfuric acid solution. The eluate is made alkaline (pH 12.) by the addition of solid barium hydroxide with stirring. The resulting barium sulfate precipitate is filtered off. The filtrate is neutralized with sulfuric acid and the precipitated barium sulfate is again filtered oil. The barium sulfate filter cake is Washed with distilled water. The filtrates are combined and concentrated by vacuum distillation to about milliliters. Crystallization of the tan-white solid occurs rapidly at room temperature (25i5 C.) on seeding the concentrate (weight, 16.1 grams). The crude product is purified by repeated recrystallizations from glacial acetic acid. A pure crystalline product having a melting point of l90-19i C. with decomposition is obtained. The White crystalline solid is soluble in water and insoluble in chloroform and ether. The pKa value is 8.6. The ultraviolet spectrum indicates a strong single peak at 278 mu, 6: 8,866 in 1 molar acid solution. The optical rotation [041 (0.5% in 0.1 N HCl solution)=+474; 0.5% in 0.1 N NaOH solution =+599; and 2.0% in distilled water:+456.

Analysiacalculated for C H O N: C, 54.20; H, 5.81; O, 31.00; N, 9.02. Found: C, 53.34; H, 6.43; O, 31.37; N, 8.86.

Example 7 A synthetic medium is prepared according to the formulation disclosed in Example 2. A 25 milliliter portion of this fermentation medium and 0.50 milliliter (2.0%) of lard oil are placed in a series of 250 milliliter Erlenmeyer flasks which are then sterilized at C. and 15 pounds per square inch pressure for 20 minutes.

The Erlenmeyer flasks are then inoculated in a separate series of fermentations, with 1.0 milliliter of inoculum of a strain of S. aureofaciens prepared according to the procedure of Example 1, i.e., Strains B740 (ATCC 12554), $609, and S77, and incubated at 27 C. for 96 hours on a rotary shaker operating at 180 revolutions per minute. The mashes are assayed spectrophotometrically and are found to contain 6-amino-5-hydroxy-L3- "2' cyclohexadienc 1 carboxylic acid in the following amounts.

Meg/ml. Strain No. 33740 (ATCC 12554) 3,900 Strain No. S609 1,800 Strain No. S77 3,700

Example 8 A fermentation medium is prepared according to the formulation disclosed in Example 4. A 25 milliliter portion of this fermentation medium and 0.625 milliliter (2.5%) of lard oil are placed in a series of 250 milliliter Erlenmeyer flasks which are then sterilized at 120 C. and 15 pounds per square inch pressure for 20 minutes. The Erlenmeyer flasks are then inoculated, in a separate series of iermentations, with 1.0 milliliter of inocuium of a strain of S. azzreofaciens prepared according to the procedure of Example 1, i.e. S652 (ATCC 13189), 5730-6, 8730-14, V11, V28 and S77, and the flasks are then incubated at 27 C. (except for Strain S77 which was incubated at 32 C.) for 96 hours on a rotary shaker operating at 180 revolutions per minute. The mashes are assayed spectrophotometrically and are found to contain 6-arnino-5-hydroxy-1,3-cyclohexadiene1-carboxylic acid in the following amounts:

Meg/ml. Strain No. S652 (ATCC 13189) 10,500 Strain No. 5730-6 2,900 Strain No. 8730-14 4,400 Strain No. V11 4,000 Strain No. V28 3,900 Strain No. S77 4,300

xample 9 6 amino--hydroxy 1,3 cyclohexadiene-l-carboxylic acid is quantitatively converted to anthranilic acid by heating (50-60 C.) with concentrated hydrochloric acid. The half-life of the reaction (t. /z) is about one hour. Anthranilic acid is identified by ultraviolet absorption spectrum, paper chromatographic evidence, and by identical melting point and mixed melting point values utilizing an anthranilic acid standard, and by comparison of its N-acetyl derivative with an authentic specimen.

eaeae Example 10 To 1.0 gram of 6-amino-5-hydroxy-1,3-cyclohexadienel-carboxylic acid is added 100 milliliters of Water and 1.0 of dehydrogenation catalyst (10% palladium on carbon). The mixture is heated at reflux for 2 hours. The hot solution is filtered. On cooling, the filtrate yields a tannish-pink crystalline solid which is separated by filtration. The solid is recrystallized twice from hot methanol, using Darco G- as a decolorizing agent; and yielding 225 milligrams of recrystallized product with a melting point of 249250 C. (with decomposition) in agreement with the literature value for 2-amino3-hydroxybenzoic acid. The ultraviolet absorption curves of 2-amino-3-hydroxybenzoic acid as given in the literature [LAOS 70, 1848 (1948)} and the product obtained by this war iple are identical.

This application is a continuation-in-part of our copending application, Serial No. 743,483, filed June 20, 1958, and now abandoned.

We claim: 1

1. T e method of producing 6-amino-5-hydroxy-1,3- eyclohexadiene-l-carboxylic acid which comprises aerobically fermenting an aqueous nutrient medium at a temperature from about 20 to 40 C. with a 6-amino-5- liydroxy-l,S-cyclohexadiene-d-carboxylic acid producing strain of S. aureofaciens, and continuing the fermentation until substantial quantities of 6-amino-5-hydroxy-1,3-cyclohexadiene l-carboxylic acid are produced.

2. The method of producing 6-amino-5-hydroxy-L3- cyclohexadiene-l-carboxylic acid which comprises aerobically fermenting an aqueous nutrient medium at a temperature from about 20 to 40 C. with a 6-amino-5- hydroxy-1,3-cyclohexadiene-1-carboxylic acid producing strain of S. aureofaciens, filtering the resulting fermented mash, acidifying the aqueous filtrate to pH 1-2, passing the aqueous filtrate through a cation exchanger, eluting the activity therefrom, and thereafter separating the activity from the eluate.

Szumski Jan. 27, 1959 McCormick et a1 Mar. 17, 1959 

1. THE METHOD OF PRODUCING 6-AMINO-5-HYDROXY-1,3CYCLOHEXADIENE-1-CARBOXYLIC ACID WHICH COMPRISES AEROBICALLY FERMENTING AN AQUEOUS NUTRIENT MEDIUM AT A TEMPERATURE FROM ABOUT 20 TO 40*C. WITH A 6-AMINO-5HYDROXY-1,3-CYCLOHEXADIENE-1-CARBOXYLIC ACID PRODUCING STRAIN OF S. AUREOFACIENS, AND CONTINUING THE FERMENTATION UNTIL SUBSTANTIAL QUANTIES OF 6-AMINO-5-HYDROXY-1,3-CYCLOHEXADIENE-1-CARBOXYLIC ACID ARE PRODUCED. 